SEARCH · Search PubMed

Results for “TRYPSINS”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

SourceResearch connectionFrom yearThrough year

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 577 records · Page 32Linked to original sources

PubMed1987

Solvent exchange of buried water and hydrogen exchange of peptide NH groups hydrogen bonded to buried waters in bovine pancreatic trypsin inhibitor.

Solvent exchange of 18O-labeled buried water in bovine pancreatic trypsin inhibitor (BPTI), trypsin, and trypsin-BPTI complex is measured by high-precision isotope ratio mass spectrometry. Buried water is labeled by equilibration of the protein in 18O-enriched water. Protein samples are then rapidly dialyzed against water of normal isotope composition by gel filtration and stored. The exchangeable 18O label eluting with the protein in 10-300 s is determined by an H2O-CO2 equilibration technique. Exchange of buried waters with solvent water is complete before 10-15 s in BPTI, trypsin, and BPTI-trypsin, as well as in lysozyme and carboxypeptidase measured as controls. When in-exchange dialysis and storage are carried out at pH greater than or equal to 2.5, trypsin-BPTI and trypsin, but not free BPTI, have the equivalent of one 18O atom that exchanges slowly (after 300 s and before several days). This oxygen is probably covalently bound to a specific site in trypsin. When in-exchange dialysis and storage are carried out at pH 1.1, the equivalent of three to seven 18O atoms per molecule is associated with the trypsin-BPTI complex, apparently due to nonspecific covalent 18O labeling of carboxyl groups at low pH. In addition to 18O exchange of buried waters, the hydrogen isotope exchange of buried NH groups H bonded to buried waters was also measured. Their base-catalyzed exchange rate constants are on the order of NH groups that in the crystal are exposed to solvent (static accessibility greater than 0) and hydrogen-bonded main chain O, and their pH min is similar to that for model compounds. The pH dependence of their exchange rate constants suggests that direct exchange with water may significantly contribute to their observed exchange rate.

Aprotinin↗

PubMed1975

Amino acid sequence of dogfish trypsin.

The amino acid sequence of pancreatic trypsin from the spiny Pacific dogfish (Squalus acanthias) has been determined and compared with the sequences of bovine and porcine trypsin. Dogfish trypsin contains one less amino acid residue (222) than the other two enzymes. Two-thirds of the residues in corresponding positions in dogfish and bovine trypsin are identical and the sequences ofall three enzymes are homologous. Of the 223 amino acid residues of bovine trypsin, 77 are replaced without significant changes in function. Seven replacements, all conservative, occur in the interior of the protein; the remainder are on the surface. All residues known to be components of the active site of bovine trypsin are present in corresponding positions in dogfish trypsin. Comparison of the three enzymes suggests calcium binding sites in dogfish trypsin. A corrected sequence of bovine trypsin identifies residue 67 as Asn and residues 84-87 as Ser-Asn-Thr-Leu.

Amino Acid Sequence↗

PubMedDate not supplied

Chemically modified, immobilized trypsin reactor with improved digestion efficiency.

Tryptic digestion followed by identification using mass spectrometry is an important step in many proteomic studies. Here, we describe the preparation of immobilized, acetylated trypsin for enhanced digestion efficacy in integrated protein analysis platforms. Complete digestion of cytochrome c was obtained with two types of modified-trypsin beads with a contact time of only 4 s, while corresponding unmodified-trypsin beads gave only incomplete digestion. The digestion rate of myoglobin, a protein known to be rather resistant to proteolysis, was not altered by acetylating trypsin and required a buffer containing 35% acetonitrile to obtain complete digestion. The use of acetylated-trypsin beads led to fewer interfering tryptic autolysis products, indicating an increased stability of this modified enzyme. Importantly, the modification did not affect trypsin's substrate specificity, as the peptide map of myoglobin was not altered upon acetylation of immobilized trypsin. Kinetic digestion experiments in solution with low-molecular-weight substrates and cytochrome c confirmed the increased catalytic efficiency (lower K(M) and higher k(cat)) and increased resistance to autolysis of trypsin upon acetylation. Enhancement of catalytic efficiency was correlated with the number of acetylations per molecule. The favorable properties of the new chemically modified trypsin reactor should make it a valuable tool in automated protein analysis systems.

Acetonitriles↗

PubMed1970

The release of radioactive nucleic acids and mucoproteins by trypsin and ethylenediaminetetra-acetate treatment of baby-hamster cells in tissue culture.

Monolayers of baby-hamster kidney cells were grown on glass in tissue culture and harvested with trypsin or EDTA in order to investigate the cell surface macromolecules removed by these cell-disaggregating agents. The release of nucleic acids from the cells during the harvesting procedure was monitored by labelling the cellular RNA with [5-(3)H]uridine and the cellular DNA with [2-(14)C]thymidine. Treatment of the cells with EDTA was found to cause an increase in the permeability of the plasma membrane with 7.6% of the cellular RNA, but less than 1% of the cellular DNA, being released. Moreover, 61% of the cells harvested with EDTA were permeable to Trypan Blue. With crude trypsin, lysis of the cell occurred with the release of similar amounts of RNA and DNA amounting to about 11% of the total cellular nucleic acid. In contrast, crystalline trypsin released only 1% of the cellular nucleic acids. Since virtually all the cells (99%) after harvesting in crystalline trypsin were impermeable to Trypan Blue, this method was suitable for obtaining cell surface macromolecules without contamination by intracellular damage. [1-(14)C]Glucosamine was incorporated by the cells only into bound hexosamines and sialic acids. [By monitoring the release of radioactivity in high-molecular-weight material in such experiments a measure of the release of macromolecules containing amino sugars was obtained.] Of the total macromolecules containing amino sugars in the cells 33%, 24% and 13% were released when the cells were harvested with crude trypsin, crystalline trypsin or EDTA respectively. Crystalline trypsin also released 39% of the total sialic acid of the cell, whereas less than 1% of the cellular sialic acid was present in the EDTA-treated fraction. It is concluded that the macromolecules containing amino sugars released with crude trypsin and EDTA are likely to be heavily contaminated with intracellular material. However, the macromolecules released by crystalline trypsin appear to come from the cell surface.

Animals↗

PubMed1975

Chemical modification of amino groups and guanidino groups of trypsin. Preparation of stable and soluble derivatives.

1. Isoionic chemical modification of amino groups of trypsin (EC 3.4.21.4) was studied for the purpose of obtaining a well-defined modified trypsin with minimum changes in physicochemical properties and with sufficient stability at neutral pH. Acetamidination with methyl acetimidate hydrochloride proceeded very rapidly at pH9.8 and 5degrees C and all 14 epsilon-amino groups were modified in 2h. The reaction was limited to epsilon-amino groups. The alpha-amino group of N-terminal isoleucine was modified only by repeated reactions in the presence of 5.5 M-guanidine or 8 M-urea. 2. The epsilon-acetamidinated derivative of beta-trypsin retained enzymic activity at values comparable with those of native enzyme tested with alpha-N-benzoyl-L-arginine ethyl ester and alpha-N-benzoyl-L-arginine p-nitroanilide as substrates; it also showed substrate activation comparable with that of native enzyme. The acetamidination of alpha-trypsin resulted in approx. 50% decrease in its esterolytic activity. 3. The epsilon-acetamidinated beta-trypsin was very stable at pH8 and 25degrees C in the absence of Ca2+. The activity of 0.04% (W/V) enzyme solution remained practically unchanged for 10h, and after 24h 90% of the activity was still retained. Possible autolytic cleavage of peptide bonds of acetamidinated enzymes was followed by N-terminal analysis by using automated Edman degradation. Only the Arg(105)-Val(106) bond was found to be cleaved to an appreciable extent. Thus beta-trypsin can be stabilized simply by complete acetamidination of epsilon-amino groups without modifying guanidino groups of arginine residues. Acetamidinated alpha-trypsin was unstable, but its inactivation at a neutral pH could not be attributed to the cleavage of a single specific peptide bond. 4. The acetamidination of the alpha-amino group of the N-terminal isoleucine results in the inactivation of esterolytic activity. However, this enzyme retained the ability to react with p-nitrophenyl p'-guanidinobenzoate. 5. It was concluded that acetamidination of beta-trypsin is a convenient method for preparing a well-defined stable and soluble trypsin derivative without appreciable change in its physical properties.

Acetamides↗

PubMed1976

The effects of proteolytic digestion by trypsin on the structure and catalytic properties of reduced nicotinamide-adenine dinucleotide dehydrogenase from bovine heart mitochondria.

1. At 21 degrees C incubation of NADH-ubiquinone-1 reductase (Complex 1) with trypsin caused selective inhibition of nicotinamide nucleotide transhydrogenase activity. The reduction of K3Fe(CN)6 by NADH or NADPH was unaffected, but a slow decrease in the rate of reduction of ubiquinone-1 by NADH was observed. 2. The pH-dependence of nicotinamide nucleotide transhydrogenase activity differed in Complex I and trypsin-treated Complex I. The trypsin-labile activity had a pH optimum of approx. 6.5, whereas the trypsin-resistant activity had a pH optimum of approx. 5.5 or less. 3. The trypsinlabile transhydrogenase activity was specifically inhibited by butanedione or phenylglyoxal and was identified with the enzyme catalysing energy-linked transhydrogenase activity in submitochondrial particles. 4. Polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate revealed that trypsin caused degradation of a polypeptide of mol.wt 20500 in parallel with the loss of transhydrogenase activity. 5. At 30 degrees C and higher trypsin concentrations, the rate of reduction of K3Fe(CN)6 by NADH or NADPH slowly decreased. Increased lability of NADH-K3Fe(CN)6 reductase activity to trypsin was observed when the endogenous phospholipid of Complex I was depleted by detergent or phospholipase A treatment. 6. Polyacrylamide-gel electrophoresis indicated that removal of phospholipid allowed much more extensive degradation of constituent polypeptides by trypsin. The subunits of the low-molecular-weight (type II) dehydrogenase (53000 and 26000 mol.wt.) were, however, relatively resistant to trypsin even in phospholipid-depleted preparations.

Animals↗

PubMed1976

A trypsin and chymotrypsin inhibitor from chick peas (Cicer arietinum).

1. A trypsin and chymotrypsin inhibitor was isolated by extraction of chick-pea meal at pH8.3, followed by (NH4)2SO4 precipitation and successive column chromatography on CM-cellulose and calcium phosphate (hydroxyapatite). 2. The inhibitor was pure by polyacrylamide-gel and cellulose acetate electrophoresis and by isoelectric focusing in polyacrylamide gels. 3. The inhibitor had a molecular weight of approx. 10000 as determined by ultracentrifugation and by polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate. A molecular weight of 8300 was resolved from its amino acid composition. 4. The inhibitor formed complexes with trypsin and chymotrypsin at molar ratios of 1:1. 5. Limited proteolysis of the inhibitor with trypsin at pH3.75 resulted in hydrolysis of a single-Lys-X-bond and in consequent loss of 85% of the trypsin inhibitory activity and 60% of the chymotrypsin inhibitory activity. Limited proteolysis of the inhibitor with chymotrypsin at pH3.75 resulted in hydrolysis of a single-Tyr-X-bond and in consequent loss of 70% of the trypsin inhibitory activity and in complete loss of the chymotrypsin inhibitory activity. 6. Cleavage of the inhibitor with CNBr followed by pepsin and consequent separation of the products on a Bio Gel P-10 column, yielded two active fragments, A and B. Fragment A inhibited trypsin but not chymotrypsin, and fragment B inhibited chymotrypsin but not trypsin. The specific trypsin inhibitory activity, on a molar ratio, of fragment A was twice that of the native inhibitor, suggesting the unmasking of another trypsin inhibitory site as a result of the cleavage. On the other hand, the specific chymotrypsin inhibitory activity of fragment B was about one-half of that of the native inhibitor, indicating the occurrence of a possible conformational change.

Amino Acids↗

PubMed1981

Characterization of a high-molecular-weight form of human acrosin. Comparison with human pancreatic trypsin.

A high-molecular-weight form of acrosin (alpha-acrosin, EC 3.4.21.10) was extracted from spermatozoa obtained from frozen semen and purified over 300-fold. Purification was effected by sequential use of Sephadex G-150, CM-cellulose and DEAE-cellulose chromatography. Properties of human acrosin were compared with those of human pancreatic trypsin. The molecular weight (Mr) of acrosin (70000) was greater than that of trypsin (Mr 21000). Isoelectric points for acrosin (pI = 9.0) and trypsin (pI = 8.2) were also different. alpha-N-Benzoyl-L-arginine ethyl ester was hydrolysed 50% more rapidly by acrosin than by trypsin. Acrosin had similar kcat. values for the hydrolysis of esters with different acylating groups (i.e. benzoyl-L-arginine and p-tosyl-L-arginine esters). In contrast, trypsin had dissimilar kcat. values for the hydrolysis of esters with different acylating groups. Kinetic data argue against deacylation as the rate-limiting step in ester hydrolysis by acrosin. Acrosin was less sensitive than trypsin to inhibition by 7-amino-1-chloro-3-L-tosylamidoheptan-2-one ('TLCK'), di-isopropyl fluorophosphate and soya-bean trypsin inhibitor. D-Fructose and D-arabinose inhibited acrosin, but had no effect on trypsin. The data indicate that definite differences exist between human acrosin and trypsin.

Acrosin↗

PubMed1997

Luminal trypsin may regulate enterocytes through proteinase-activated receptor 2.

Proteinase-activated receptor 2 (PAR-2) is a recently characterized G-protein coupled receptor that is cleaved and activated by pancreatic trypsin. Trypsin is usually considered a digestive enzyme in the intestinal lumen. We examined the hypothesis that trypsin, at concentrations normally present in the lumen of the small intestine, is also a signaling molecule that specifically regulates enterocytes by activating PAR-2. PAR-2 mRNA was highly expressed in the mucosa of the small intestine and in an enterocyte cell line. Immunoreactive PAR-2 was detected at the apical membrane of enterocytes, where it could be cleaved by luminal trypsin. Physiological concentrations of pancreatic trypsin and a peptide corresponding to the tethered ligand of PAR-2, which is exposed by trypsin cleavage, stimulated generation of inositol 1,4,5-trisphosphate, arachidonic acid release, and secretion of prostaglandin E2 and F1alpha from enterocytes and a transfected cell line. Application of trypsin to the apical membrane of enterocytes and to the mucosal surface of everted sacs of jejunum also stimulated prostaglandin E2 secretion. Thus, luminal trypsin activates PAR-2 at the apical membrane of enterocytes to stimulate secretion of eicosanoids, which regulate multiple cell types in a paracrine and autocrine manner. We conclude that trypsin is a signaling molecule that specifically regulates enterocytes by triggering PAR-2.

Animals↗

PubMed1997

Characterization of the secondary structure and assembly of the transmembrane domains of trypsinized Na,K-ATPase by Fourier transform infrared spectroscopy.

Fourier transform infrared spectroscopy has been used to compare native Na,K-ATPase-containing membranes with those trypsinized in the presence of either Rb+ or Na+ ions to remove the extramembranous parts of the protein. The protein secondary structure content deduced from the amide I band is approximately 30-35% alpha-helix, 37-40% beta-structure, and 13-15% random coil for native membranes from shark rectal gland and from pig kidney, in both the Na- and K-forms. Trypsinization in either Rb+ (a K+ congener) or Na+ removes approximately 35% of the amide I band intensity of native membranes from shark rectal gland. The protein secondary structural content of the trypsinized membranes lies in the range of approximately 23-32% alpha-helix, 37-46% beta-structure, and 12-18% random coil for the shark and kidney enzymes. The distribution of intensity between the bands corresponding to protonated and deuterium-exchanged alpha-helices, and between the component bands attributed to beta-structure, changes considerably on trypsinization, in the direction of a greater proportion of protonated alpha-helix and a broader range of frequencies for beta-structure. The kinetics of deuteration of the slowly exchanging population of protein amide groups is also changed on trypsinization. The mean rate constant for deuteration of trypsinized membranes is approximately half that for native membranes, whereas the proportion of amides contributing to this population increases on trypsinization. The temperature dependence of the amide I band in the Fourier transform infrared spectra indicates that the onset of thermal denaturation occurs at 58 degrees C for native membranes (in either Na+ or K+) and for membranes trypsinized in Rb+, but the major denaturation event for membranes trypsinized in Na+ occurs at approximately 84 degrees C. These results correlate with the functional properties of the intramembranous section of the enzyme.

Animals↗

PubMed2004

Trypsin IV, a novel agonist of protease-activated receptors 2 and 4.

Certain serine proteases signal to cells by cleaving protease-activated receptors (PARs) and thereby regulate hemostasis, inflammation, pain and healing. However, in many tissues the proteases that activate PARs are unknown. Although pancreatic trypsin may be a physiological agonist of PAR(2) and PAR(4) in the small intestine and pancreas, these receptors are expressed by cells not normally exposed pancreatic trypsin. We investigated whether extrapancreatic forms of trypsin are PAR agonists. Epithelial cells lines from prostate, colon, and airway and human colonic mucosa expressed mRNA encoding PAR(2), trypsinogen IV, and enteropeptidase, which activates the zymogen. Immunoreactive trypsinogen IV was detected in vesicles in these cells. Trypsinogen IV was cloned from PC-3 cells and expressed in CHO cells, where it was also localized to cytoplasmic vesicles. We expressed trypsinogen IV with an N-terminal Igkappa signal peptide to direct constitutive secretion and allow enzymatic characterization. Treatment of conditioned medium with enteropeptidase reduced the apparent molecular mass of trypsinogen IV from 36 to 30 kDa and generated enzymatic activity, consistent with formation of trypsin IV. In contrast to pancreatic trypsin, trypsin IV was completely resistant to inhibition by polypeptide inhibitors. Exposure of cell lines expressing PAR(2) and PAR(4) to trypsin IV increased [Ca(2+)](i) and strongly desensitized cells to PAR agonists, whereas there were no responses in cells lacking these receptors. Thus, trypsin IV is a potential agonist of PAR(2) and PAR(4) in epithelial tissues where its resistance to endogenous trypsin inhibitors may permit prolonged signaling.

Animals↗

PubMed1983

Amiloride-sensitive trypsinization of apical sodium channels. Analysis of hormonal regulation of sodium transport in toad bladder.

Incubation of the mucosal surface of the toad urinary bladder with trypsin (1 mg/ml) irreversibly decreased the short-circuit current to 50% of the initial value. This decrease was accompanied by a proportionate decrease in apical Na permeability, estimated from the change in amiloride-sensitive resistance in depolarized preparations. In contrast, the paracellular resistance was unaffected by trypsinization. Amiloride, a specific blocker of the apical Na channels, prevented inactivation by trypsin. Inhibition of Na transport by substitution of mucosal Na, however, had no effect on the response to trypsin. Trypsinization of the apical membrane was also used to study regulation of Na transport by anti-diuretic hormone (ADH) and aldosterone. Prior exposure of the apical surface to trypsin did not reduce the response to ADH, which indicates that the ADH-induced Na channels were inaccessible to trypsin before addition of the hormone. On the other hand, stimulation of short-circuit current by aldosterone or pyruvate (added to substrate-depleted, aldosterone-repleted bladders) was substantially reduced by prior trypsinization of the apical surface. Thus, the increase in apical Na permeability elicited by aldosterone or substrate involves activation of Na channels that are continuously present in the apical membrane in nonconductive but trypsin-sensitive forms.

Aldosterone↗

PubMed1992

Insensitivity of guinea pig ventricular delayed rectifier IK to intracellular trypsin: implications for channel structure and function.

OBJECTIVE: Intracellular application of proteolytic agents modifies the function of many voltage gated ion channels. The presence of a trypsin sensitive inhibitory domain in a channel protein may be important for G protein dependent activation. Guinea pig ventricular IK is modulated by a direct G protein pathway. The aim was to determine if guinea pig ventricular IK is also modified by intracellularly applied trypsin. METHODS: Whole cell and excised inside out configurations of patch clamp were used to record IK from guinea pig ventricular myocytes before and after cytosolic application of trypsin (1 mg.ml-1). We used previously reported effects of trypsin on the L type calcium current (ICa) to monitor dialysis time and enzyme activity in whole cell experiments where IK and ICa were measured concomitantly. RESULTS: Addition of trypsin to the solution bathing the cytosolic face of excised membrane patches had no effect on the amplitude or kinetics of IK. When added to the pipette solution and introduced by cell dialysis, trypsin had no effect on whole cell IK, even when significant effects on the amplitude and kinetics of ICa were evident. CONCLUSIONS: Guinea pig ventricular IK is not enhanced or otherwise altered by intracellularly applied trypsin. Therefore direct phosphorylation independent enhancement of IK by guanine nucleotides cannot depend on interactions between G protein subunits and trypsin sensitive inhibitory channel domains. The lack of trypsin modification of cardiac ventricular IK suggests that the structure of the endogenous delayed rectifier K+ channel may be different than that of other voltage gated channels.

Animals↗

PubMed1998

Differential expression of urinary inter-alpha-trypsin inhibitor trimers and dimers in normal compared to active calcium oxalate stone forming men.

PURPOSE: We determine if the immunoreactive profile of urinary inter-alpha-trypsin inhibitor can be used to distinguish between normal individuals and individuals with calcium oxalate stone disease. MATERIALS AND METHODS: Urinary proteins were dialyzed against water (15 kDa. molecular weight cutoff), lyophilized and resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (6% acrylamide, reducing conditions) followed by Western blot. Inter-alpha-trypsin immunoreactive proteins were detected by enhanced chemiluminescence. Stone formation was confirmed to be active radiologically or passed as stone or gravel within 12 months of the sample. Stone composition was confirmed crystallographically. Normal individuals had no personal or familial history of urolithiasis and matched stone forming patients regarding race (white) and age (23 to 71 years old). Urine from a total of 101 individuals was analyzed. RESULTS: The intact inter-alpha-trypsin trimer (approximately 220 to 240 kDa.) and heavy chain (HC) 2-bikunin/HC1-bikunin dimers (approximately 115 to 130 kDa.) were detected more often in stone forming men (23 of 26 [89%] and 26 of 26 [100%], respectively) than in normal individuals (6 of 26 [23%] and 5 of 26 [19%], respectively, p < 0.0001). In those normal individuals who expressed inter-alpha-trypsin trimer and HC-bikunins the relative intensities were 5.3+/-1.4% and 16.3+/-17.1% of the stone forming controls, respectively. The identity of high molecular weight-inter-alpha-trypsin immunoreactive bands was confirmed using antibodies against the individual subunits (HC1, HC2, HC3, bikunin). In contrast to men high molecular weight-inter-alpha-trypsin's were readily detected in normal and stone forming women with equal frequency (inter-alpha-trypsin-trimer p=0.1337, HC-bikunins p=0.2836): inter-alpha-trypsin-trimer 17 of 18 [94%] and 9 of 13 [77%]; HC-bikunins 17 of 18 [94%] and 10 of 13 [85%]). Inter-alpha-trypsin-trimer and HC-bikunins, respectively, were detected in 2 and 5 of 10 patients with chronic renal disease. Expression was not related to hematuria or proteinuria. CONCLUSIONS: Immunoreactive profiles of urinary proteins may be able to be developed into a useful diagnostic tool to identify active stone formation, although a separate panel may be required for men and women. It is possible that these differences may provide clues as to why the incidence of stone disease is higher in men than women.

Adult↗

PubMed1991

Urinary levels of activated trypsin in whole-organ pancreas transplant patients with duodenocystostomies.

Urinary levels of trypsinogen and active trypsin were measured in 34 urine samples from 14 patients an average of 23 months following whole organ pancreas transplantation and duodenocystostomy. Timed urine specimens obtained 30 min and 90 min following a time zero void were collected from 5 patients in an effort to define the kinetics of trypsinogen secretion and activation. Total urinary protein and urinary pH were correlated with urinary levels of trypsin and trypsinogen. Twenty-one specimens from 8 normal volunteers and a single specimen from a pancreas transplant patient with a duodenoenterostomy served as controls. Activated trypsin was present in 33 of 34 specimens from 13 of the 14 transplant patients. The average total trypsin activity in all samples was 84.4 micrograms/ml urine (+/- SE 9.6). Trypsinogen was present in 13 of 34 samples, from 7 of 14 patients. The average trypsinogen concentration of all 34 samples was 9.6 +/- 6.2 micrograms/ml. No trypsin or trypsinogen activity was identified in any control sample. In the 5 patients undergoing timed urine collections total trypsin increased an average of 1.3-fold at 30 min and 1.1-fold at 90 min relative to time zero. Urinary trypsinogen increased an average of 7.1-fold at 30 min and 3.1-fold at 90 min following the initial void. Urinary pH and total urinary protein failed to show a significant correlation with urinary levels of total trypsin or trypsinogen. These data suggest that trypsinogen is rapidly converted to active trypsin following secretion into the bladder, resulting in the high urinary trypsin levels that were detected in the majority of patients.

Cystostomy↗

PubMed1981

The influence of effectors on the refolding (reactivation) of immobilized trypsin.

The refolding of immobilized trypsin in the presence of various effectors of its enzymatic activity has been studied. Trypsin covalently bound to Sephadex G-200 was made to unfold in a concentrated solution of urea; at the same time its S-S bonds were split with the help of dithiothreitol. The preparation was then separated from the splitting agents, and one of the effectors of the enzymatic activity of trypsin (boric acid, benzamidine, pancreatic or soybean inhibitors of trypsin, N-benzoyl-L-arginine ethyl ester, and N-tosyl-L-arginine methyl ester) was added and the reactivation of immobilized enzyme was studied in the absence of catalysts of thiol-disulphide exchange. The following effects were found. 1. The reactivation of immobilized trypsin in the presence of specific substrates or protein inhibitors proceeds with the same yield (2-5%) as in their absence. 2. In the presence of benzamidine or boric acid (competitive inhibitors) the reactivation yields of the immobilized trypsin increased 5-fold and 12-fold respectively, and became equal to 15% and 40%. Comparison of these results with the statistical probability of formation of the six native S-S bonds from twelve SH groups (approximately 0.01%) shows that if trypsin is made to refold in an immobilized state in the presence of a 'good' effector, the yield of the reactivation of the enzyme can be increased several thousand times. 3. Similar effects were observed for trypsin immobilized on Sepharose 4B. A model is suggested in terms of which the influence of various effectors on trypsin refolding is explained as being a result of their ability to bind with an intermediate folded forms of protein followed by a shift of the equilibrium towards 'regular' conformers.

Catalysis↗

PubMed2002

Modified proteinase-activated receptor-1 and -2 derived peptides inhibit proteinase-activated receptor-2 activation by trypsin.

Trypsin activates proteinase-activated receptor-2 (PAR(2)) by a mechanism that involves the release of a tethered receptor-activating sequence. We have identified two peptides, FSLLRY-NH(2) (FSY-NH(2)) and LSIGRL-NH(2) (LS-NH(2)) that block the ability of trypsin to activate PAR(2) either in PAR(2)-expressing Kirsten virus-transformed kidney (KNRK) cell lines or in a rat aorta ring preparation. The reverse PAR(2) peptide, LRGILS-NH(2) (LRG-NH(2)) did not do so and FSY-NH(2) failed to block thrombin activation of PAR(1) in the aorta ring or in PAR(1)-expressing human embryonic kidney cells. Half-maximal inhibition (IC(50)) by FSY-NH(2) and LS-NH(2) of the activation of PAR(2) by trypsin in a PAR(2) KNRK calcium-signaling assay was observed at about 50 and 200 microM, respectively. In contrast, the activation of PAR(2) by the PAR(2)-activating peptide, SLIGRL-NH(2) (SL-NH(2)) was not inhibited by FSY-NH(2), LS-NH(2), or LRG-NH(2). In a casein proteolysis assay, neither FSY-NH(2) nor LS-NH(2) inhibited the proteolytic action of trypsin on its substrate. In addition, FSY-NH(2) and LS-NH(2) were unable to prevent trypsin from hydrolyzing a 20-amino acid peptide, GPNSKGR/SLIGRLDTPYGGC representing the trypsin cleavage/activation site of rat PAR(2). Similarly, FSY-NH(2) and LS-NH(2) failed to block the ability of trypsin to release the PAR(2) N-terminal epitope that is cleaved from the receptor upon proteolytic activation of receptor-expressing KNRK cells. We conclude that the peptides FSY-NH(2) and LS-NH(2) block the ability of trypsin to activate PAR(2) by a mechanism that does not involve a simple inhibition of trypsin proteolytic activity, but possibly by interacting with a tethered ligand receptor-docking site.

Animals↗

PubMed1987

Activation of botulinum C2 toxin by trypsin.

C2 toxin (C2T) elaborated by Clostridium botulinum types C and D is composed of two dissimilar protein components, designated components I and II. The biological activity of C2T is enhanced by treating the toxin with trypsin. This activation of C2T is observed as a result of mixing untrypsinized component I and trypsinized component II but not as a result of mixing trypsinized component I and untrypsinized component II. The data presented here show that the maximum lethality of C2T, determined by mixing untrypsinized component I and trypsinized component II, was attained by treating component II with trypsin at a ratio of 10:1 on a protein basis for 30 min at 35 degrees C at pH 7.5. The activation of component II was always accompanied by a change in the molecular weight of the component from 101,000 to 88,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). However, the gel filtration of trypsinized component II resulted in the separation of two active components, with apparent molecular weights, estimated from the elution volume by gel filtration, of 365,000 and 74,000. The high-molecular-weight component II had hemagglutination and hemolytic activities, whereas the low-molecular-weight component II has only hemagglutination activity. These two molecular species of active component II had approximately the same lethality, when mixed with component I, and gave a single band in SDS-PAGE, with a molecular weight of 88,000, the same as that of trypsin-activated component II under different reaction conditions. The results indicate that the activation of C2T by trypsin is due to the molecular conversion of component II from molecular weight 101,000 to 88,000 as determined by SDS-PAGE and that the trypsin-activated component II tends to form an oligomer of the active component II.

Animals↗